In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detection light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the path of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To make possible acquisition of image data in layers, the sample stage or the objective is shifted after a layer is scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
German Unexamined Application DE 199 54 933 A1 discloses an arrangement for incoupling at least one beam or an optical tweezers for grasping particles, and/or for incoupling a processing beam, into a microscope beam path, preferably in a laser scanning microscope, means being provided for freely adjustable modification of the location of the beam focus of the optical tweezers and/or the processing beam in terms of modification of the focus position of the microscope. In this arrangement, the focus of the processing beam or optical tweezers is displaceable in the Z direction by displacement of an optical system in the beam path of the processing beam or optical tweezers. Displacement of the focus in the X/Y direction is possible only by displacement of the specimen stage. This is disadvantageous for the user because displacement of the focus is necessarily associated with a change in the image area. In addition, rapid movement of the focus is not possible.
German Application DE 100 39 520 A1 discloses an apparatus and a method for the examination and manipulation of microscopic specimens, having a microscope, a light source serving to illuminate the specimen, an illumination beam path, a detector serving to detect the light returning from the specimen, a detection beam path, a light source serving for specimen manipulation, and a manipulating light beam path. The apparatus and the method according to the invention make possible three-dimensional examination and manipulation of specimens whose extension along the optical axis is greater than the depth-of-field range of the microscope objective being used, specimen manipulation also being said to be possible at all points of the three-dimensional specimen. Three-dimensional detection of the specimen in which a discrimination is performed of specimen light contributions coming from regions that lie beyond the depth-of-field range of the microscope objective is also said to be possible. The apparatus and the method according to the invention are characterized in that the microscope is a confocal scanning microscope. The apparatus contains a beam deflection device for guiding the illuminating light beam and a further one for guiding the manipulating light beam, in which context provision can be made for the beam deflection apparatuses to operate synchronously with one another. The apparatus is therefore very flexible and moreover permits rapid movement of the manipulating light beam. The apparatus is, however, very costly and complex in terms of construction and operation.